Experimental Study On The Differentiation And Mechanism Of Rat Bone Marrow Stromal Stem Cells Into Hepatocytes

Objective Bone marrow stromal stem cells (BMSCs) arise from the complex array of supportingstructures in marrow. BMSCs can be isolated easily from bone marrow, and they can beexpanded with high efficiency, and induced to differentiate to multiple lineages under defined cultureconditions. These cells have generated a great deal of interest because BMSCs have a highproliferative potential in culture, also they are capable of differentiating to a wide variety of cell types,and when autologous transplanted, there exist no immunorejection. So BMSCS have great potentialuse in regenerative medicine and tissue engineering like treating hepar dieases. We studied theisolation, culture and induced differentiation of rat BMSCs, especially the proper condition andmechanism of hepatocytes differentiation in vitro and in vivo, and identified the hepatocytes toprovide an experimental groundwork for the therapy of hepatic diseases using transplantion.Methods BMSCs were isolated from bone marrow of rats, then cultured in low-glucose DMEM with20% NCS for expanding, and observed the growth and biology characterization of BMSCs. Thegrowth curve of cell proliferation was obtained by the MTI" method; Based on the proliferationstatus of BMSCs we provided them cell factor hepatocyte abstraction(G 200μg/ml, 500μg/ml),β-NGFfrom rat(20ng/ml, 50ng/ml),β-NGF from human (20ng/ml, 50ng/ml) and (50μg/ml) and comparedwith the control group; RT-PCR and immunocytochemistry were used to detect the exclusive markerof hepatocytes albumin (ALB) and alpha antitrypsin (AAT) respectively, We also identified thehepatocytes using ICG, and proved it having the function of hepatocytes; We injected BMSCs labelledwith BrdU into the tail vein of mice to test by immunohistochemistry whether BMSCs coulddifferentiate into hepatocytes and express hepatocellular phenotypes in vivo. The result of serologyshowed stem cells'effect of curing hepatic injury.Results (1) Based on the growth curve of cell proliferation, BMSCs of 3rd-5th generation werevery purified and all in the whirlpool-shape. They proliferated more rapidly than those of 8th and 10thgeneration. BMSCs were low-differentiation under t he electron microscopy. The method ofimmunocytochemistry revealed that G,β-NGF from rat,β-NGF from human and HGF all expressedalpha-fetoprotein compared with control groups(P＜0.05). The expression of AAT was more in G,β-NGF group than HGF(P＜0.05). (3) IRT-PCR showed that oval cells induced by these four cell factorall express albumin mRNA and they could ingest ICG and took on green. (4) There exist transplantedcells labelled with BrdU in the liver of recipient mice after being transplanted 7d, 14d and 28d. Thetransplanted BMSCs distribute in the liver parenchama. The result of serology showed that BMSCs could treat the hepatic injury effectively after after being transplanted14d and 28d compared with control group(P＜0.05). (5) [Ca²⁺] i of hepatocytes we induced washigher than control group(P＜0.05) and nimodipine could block BMSCs differential into hepatocytes.Conclusion (1) BMSCs of 3rd -5th generation were very purified and proliferated more rapidly thanother generation, The cytokine G,β-NGF from rat,β-NGF from human and HGF could induceBMSCs into hepatocytes under special condition, the microenvironment and the cytokine hadimportant effect on the differentiation of BMSCs. (3) Ca²⁺ played significant role on thedifferentiation of BMSCs. (4) The transplanted BMSCs can differentiate into hepatocytes, and havethe function of curing the hepatic injury.